Using a combination of approaches, including gene-disrupted mice, expression profiling, and pharmacology in vitro and in vivo, we recently made the surprising discovery that the tryptophan hydroxylase (TPH) gene is an important regulator of mammary gland function. TPH catalyzes the conversion of L-tryptophan to 5-hydroxytryptophan, which is the rate-limiting substrate for serotonin (5-HT) synthesis. We showed that TPH is induced by milk stasis, and that 5-HT participates in an autocrine/paracrine feedback loop that inhibits lactation. These discoveries imply that biogenic monoamines (esp. 5-HT) are important mammary-derived signaling molecules. The central objective of our plan is to explore critical details of the mechanisms by which 5-HT acts within the mammary gland (aims 1-3). A subsidiary objective (aim 4) is to determine whether 5-HT is exported from the mammary gland into the maternal circulation and/or milk. The situation we now confront is that we know neither what specific aspects of mammary physiology and development are influenced by 5-HT, nor do we know the pharmacological profile (receptor types) for the 5-HT regulated functions in the mammary glands. To fill these knowledge gaps we will make use of the wealth of available serotonergic agents to examine mammary gene regulation, proliferation, and apoptosis in organotypic cultures and primary cultures of dissociated cells. The means to do in vivo studies of mammary TPH functions are very limited because of the confounding effects that pharmacological agents have on tissues other than the mammary gland (esp. the brain). To circumvent these problems we need genetic models in which to do the physiological and developmental studies, but useful models, such as TPH gene knockouts, have not been made by other labs. Consequently, we propose to make mice in which the TPH gene is disrupted in the mammary glands, and transgenic mice expressing the human serotonin transporter (hSERT) in the mammary glands. These models will allow us to examine the role of mammary-expressed TPH in development and physiology in animals that have interruptions in their autocrine/paracrine 5-HT exposure.